Biophysical characterization of human platelet profilin

Abstract

Cell division, motility, morphology and membrane trafficking are essential physiological processes for maintaining cellular homeostasis. In eukaryotic cells, cytoskeletal regulation is controlled by a number of actin modulating proteins that localize, nucleate, bundle, crosslink, cap, sever, sequester and recycle the cytoplasmic and membrane associated actin microfilament networks. Human profilin is an actin monomer (G-actin) binding protein that is thought to localize and release G-actin at the plasma membrane, making available a pool of polymerization competent actin necessary for filament elongation, branch formation, protrusion and ultimately cell locomotion. One model of cell motility proposes that profilin shuttles G-actin to the membrane periphery, where the profilin/G-actin complex is disrupted by membrane associated polyphosphoinositides thereby increasing the pool of membrane-proximal polymerization competent G-actin.;The mechanism of G-actin release has not been elucidated. The prevailing paradigm holds that G-actin release at the cell cortex is due to competition between PIP2 and G-actin for overlapping binding sites on profilin. My hypothesis is that the acidic phospholipid PIP2 introduces structural changes in profilin, leading to a reduction in conformational stability and partial unfolding of the polypeptide chain. I propose that this unfolding event is central to actin release in vivo. In addition to direct phospholipid interactions, the lower-than-bulk pH immediately proximal to the plasma membrane may play an important role in the regulation of profilin structure and function. The overall goal of my thesis research is to identify and characterize the structural, energetic and dynamic alterations in human platelet profilin (Hpp) that are associated with PIP2 binding and to extend these findings to develop a mechanism that describes the structural basis for G-actin release at the plasma membrane. To achieve this goal the biophysical behavior of isolated Hpp and Hpp in complex with PIP2 micelles, poly-L-proline and G-actin has been studied by circular dichroism, tryptophan fluorescence and nuclear magnetic resonance spectroscopy.;Acid titration of Hpp identified a stable equilibrium folding intermediate that is maximally populated between pH 4.0 and pH 5.0 (i.e. N ⇒ I ⇒ U). The physical properties of this equilibrium intermediate are near native; however, the conformational stability is reduced by 50%. (Abstract shortened by UMI.).